PROJECT SUMMARY/ABSTRACT Significant bone loss and defective bone healing in age-related bone diseases cause severe pain and high morbidity and mortality, and are a major health issue in the U.S. Lifelong regeneration of bone and cartilage requires skeletal stem cells (SSCs), and a skeletal stem/progenitor cell subset in periosteum (P-SSCs) has been known to play an important role for bone repair. However, due to the wide tissue distribution of SSCs and the lack of specific markers to distinguish rare P-SSCs in vivo, how endogenous P-SSCs respond to bone injury and how they function in bone healing are unknown. Therefore, the goal of our proposal is to define the in vivo identity and function of P-SSCs and the mechanisms that regulate P-SSCs in order to control bone regeneration and repair under physiologic circumstances. We will also explore the induction of endogenous P- SSC migration and proliferation with the goal of improving recovery from age-related bone injuries. Using newly generated reporter mouse models in combination with intravital imaging technology, we recently determined that a combination of SSC markers, myxovirus resistance-1 (Mx1) and alpha smooth muscle actin (?SMAGFP) can selectively label endogenous P-SSCs that are exclusively present in the periosteal cambial layer with Prx1GFP expression. In addition, sequential in vivo imaging revealed that Mx1 ?SMAGFP P-SSCs, rather than BM-SSCs, rapidly respond to the injury and continually supply new osteoblasts for injury repair in vivo. These P-SSCs show higher expression of CCR3/CCR5, the receptors for chemokine (C-C motif) ligand 5 (CCL5), than BM-SSCs and other bone cells. Moreover, we found for the first time that periosteal administration of CCL5 stimulates the real-time migration of these P-SSCs toward injury sites in vivo. Human primary periosteal cells also express CCR5 and conduct CCL5-mediated migration. We thus hypothesize that P-SSCs and their migration, upon CCL5 signaling, are necessary for bone repair, and that exogenous CCL5 provision improves the healing of age-related bone defects. Our novel intravital imaging technology and the various animal models enable us to track endogenous stem cells present in the periosteum and bone marrow as well as their differentiation at the single-cell level in living animals. We thus plan to pursue the following specific aims. In aim 1, we will determine whether inflammatory stimuli such as CCL5 are necessary for P-SSC migration and bone healing by generating a conditional ablation of CCL5 in immune cells. We will also examine if CCL5 is specific for P-SSC migration. In aim 2, we will determine whether P-SSCs and their migration are required for injury repair by performing local ablation of P-SSCs and by generating a conditional deletion of the CCR5 in P-SSCs. In aim 3, we will determine whether the local provision of exogenous CCL5 induces the migration and activation of endogenous P-SSCs early in the repair process, leading to accelerated and improved bone healing in aged mice. Upon completion of this work, we will achieve new biological insights into periosteal SSCs and will define new therapeutic targets for reversing bone diseases and defects.